Phospholipase C-gamma1 (PLC-gamma1) plays a critical role in calcium induced differentiation in part because of its ability to maintain high levels of intracellular calcium (Cai) through the provision of two important second messengers, inositol tris phosphate (IP3) and diacyl glycerol (DG). IP3 stimulates the release of calcium from intracellular stores by activating its receptor IP3R, a calcium channel in the endoplasmic reticulum and Golgi, whereas DG activates the classic and novel protein kinases C. Preliminary findings from our laboratory have demonstrated two pathways mediating calcium-induced activation of PLC-gamma1 in normal keratinocytes: one via src kinases and the other via phosphatidylinositol 3 kinases (PI3K). Furthermore, in normal keratinocytes much of the PLC-gamma1 colocalizes in the cell with other members of the calcium-signaling pathway including the calcium receptor (CaR), the IP3 receptor (IP3R), and the Golgi calcium pump (ATP2C1) suggesting an intimate interaction between PLC-gamma1 and the molecular regulators of intracellular calcium levels. Accordingly we propose the following hypothesis: PLC-gamma1 plays a central role in the mechanism by which calcium induces keratinocyte differentiation. Calcium activates PLC-gamma1 through src kinases and PI3K and promotes its interaction with regulators of Cai stores such as the IP3R and Golgi calcium pump (ATP2C1) that provide for the sustained increase in Cai essential for differentiation. To test this hypothesis we propose 4 specific aims. In the first two aims, we will explore the mechanisms by which calcium activates PLC-gamma1 in normal cells. In the third aim we will examine whether PLC-gamma1 activation by src kinases and PI3K is required for its interaction with IP3R and ATP2C1. In these aims we will compare the effects of calcium to those of EGF, which also activates PLC-gamma1 but stimulates proliferation rather than differentiation, with the expectation of finding differences between the two with respect to the mechanism of PLC-gamma1 activation. Because differentiation in vitro does not achieve the final product, namely the formation of a competent permeability barrier, in the fourth aim we will develop an in vivo model to analyze the impact of selectively deleting PLC-gamma1 expression with respect to the program of differentiation leading up to the development and regulation of the permeability barrier. Calcium signaling and calcium induced differentiation in the keratinocytes from these animals will also be studied in vitro to evaluate the comparability with earlier studies of human keratinocytes in which the PLC-gamma1 gene was knocked out by antisense techniques. These studies will increase our understanding of keratinocyte differentiation and may provide therapeutic targets for diseases in which epidermal differentiation is abnormal.